Zinc is an essential nutrient because of the important roles this metal plays as a catalytic and structural cofactor. Several zinc-dependent proteins reside within organelles such as the endoplasmic reticulum (ER), Golgi, and mitochondria. Therefore, transporter proteins are needed to distribute zinc into intracellular compartments. Because excess zinc can be toxic, cells also require homeostatic mechanisms to control the intracellular levels of free or labile zinc in the cytosol and within organelles. Cellular zinc homeostasis is achieved by several mechanisms including the control of zinc uptake, efflux, vesicular storage, and binding by metallothionein. In this proposal, another important facet of zinc homeostasis is considered, i.e. the control of zinc levels within intracellular organelles. These processes will be studied using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Preliminary results have raised five central hypotheses that provide the foundation for the specific aims of this proposal: 1) The yeast Msc2 and Zrg17 proteins form a heteromeric complex that transports zinc into the ER. In Aim 1, the composition of this complex will be assessed and its zinc transport activity will be characterized. 2) It is proposed that zinc finger domains will make useful probes of zinc status in the ER and other compartments of living cells. In Aim 2, new in vivo zinc sensors will be developed based on fluorescence resonance energy transfer (FRET). These and other assays to be developed will be used to define the role of Msc2/Zrg17 and other transporters in maintaining ER zinc homeostasis. 3) Zinc transport into the ER via the Msc2/Zrg17 complex is regulated in response to zinc status by both transcriptional and post-translational control mechanisms. In Aim 3, the molecular mechanisms of these regulatory systems will be dissected and their roles in maintaining ER zinc status determined. 4) It is proposed that zinc transport into the ER is mediated by transporters in addition to the Msc2/Zrg17 complex. In Aim 4, other zinc transporters for this compartment will be identified. 5) Several studies of mammalian cells showed that zinc deficiency leads to increased oxidative stress, the source of which has long been a mystery. In Aim 5, the hypothesis that the oxidative stress of zinc deficiency arises from the loss of ER zinc homeostasis will be tested. These aims represent a cohesive and comprehensive analysis of zinc homeostasis in the secretory pathway of eukaryotic cells.7.